Morphologic, phenotypic, and genotypic similarities between primary tumors and corresponding 3D cell cultures grown in a repeatable system-preliminary results.

BACKGROUND: Three-dimensional (3D) cell cultures are the new frontier for reproducing the tumor micro-environment in vitro. The aims of the study were (1) to establish primary 3D cell cultures from canine spontaneous neoplasms and (2) to demonstrate the morphological, phenotypic and genotypic similarities between the primary canine neoplasms and the corresponding 3D cultures, through the expression of tumor differentiation markers. RESULTS: Seven primary tumors were collected, including 4 carcinomas and 3 soft tissue sarcomas. 3D cell cultures reproduced the morphological features of the primary tumors and showed an overlapping immunophenotype of the primary epithelial tumors. Immunohistochemistry demonstrated the growth of stromal cells and macrophages admixed with the neoplastic epithelial component, reproducing the tumor microenvironment. Mesenchymal 3D cultures reproduced the immunophenotype of the primary tumor completely in 2 out of 3 examined cases while a discordant expression was documented for a single marker in one case. No single nucleotide variants or small indel were detected in TP53 or MDM2 genes, both in primary tumors and in 3D cell cultures specimens. In one sample, MDM2 amplicons were preferentially increased in number compared to TP53 ones, indicating amplification of MDM2, detectable both in the primary tumor and in the corresponding cell culture specimen. CONCLUSION: Here we demonstrate a good cell morphology, phenotype and genetic profile overlap between primary tumors and the corresponding 3D cultures grown in a repeatable system.

Longitudinal lymph node step-sectioning for the identification of metastatic disease in canine mast cell tumor.

Lymph node (LN) metastasis in canine mast cell tumor (MCT) can affect prognosis and postsurgical treatment recommendations; however, routine histological single-section examination may underestimate the incidence of metastases. This prospective study aimed at determining whether longitudinal step-sectioning of the entire LN allows for a more reliable detection of metastases. Dogs with MCT undergoing resection of the primary tumor and regional lymphadenectomy were enrolled. Formalin-fixed LNs were bisected longitudinally, both halves were embedded in paraffin and histological sections prepared at 200 µm steps. The nodal mast cells were classified according to the Weishaar classification. First-section evaluation (FSE; ie, examination of the first section obtained from the blocks) and whole LN step-section evaluation (SSE) were compared. Fifty-eight LNs were included. The median number of sections per LN was 6 (range, 3-28). FSE with toluidine blue (TB) revealed 27 (47%) nonmetastatic (HN0), 14 (24%) premetastatic (HN1), 9 (15%) early metastatic (HN2), and 8 (14%) overtly metastatic (HN3) LNs. SSE with TB resulted in upgrading the LN status in 2 cases (HN2 to HN3; HN0 to HN1). Evaluation of the first section plus an additional step-section resulted in 100% accuracy. Compared with SSE with TB, the accuracy of FSE with HE was 98% for HN3 LNs and 74% for HN2 LNs. FSE appears to reliably allow for the detection of LN metastasis in MCT, although examination of a further parallel section at a 200 µm step may increase the accuracy. A metachromatic stain is recommended for the identification of early metastases.